A widely used mechanism of gene regulation in bacteria is a noncoding RNA element called a riboswitch. These are cis-acting elements found in the leader sequence of mRNAs and regulate gene expression through their ability to directly bind a specific effector molecule to a highly structured aptamer domain. Effector binding to the aptamer domain is communicated to a downstream secondary structural switch in the expression platform that interfaces with the expression machinery (either RNA polymerase or the ribosome). In a broad spectrum of bacteria, particularly Firmicutes and Fusobacteria, central metabolic pathways including purine, amino acid, and cofactor biosynthesis and transport are regulated by riboswitches. Furthermore, genes essential for survival or virulence are under riboswitch control in a number of medically important pathogens including Listeria monocytogenes, Staphylococcus aureus, Pseudomonas aeruginosa, Clostridium difficile, and Mycobacterium tuberculosis making them of great interest as novel targets for antimicrobial therapeutics. In addition, riboswitches serve as powerful model systems for understanding various aspects of RNA biology including structure, folding and mechanisms of regulatory activity along with developing tools and methodologies for designing small molecules that target other RNAs of medical interest. Towards the long-term goal of developing a molecular understanding of how RNA interacts with small molecules and the mechanisms it uses to regulate gene expression, we are using purine- and cobalamin- binding riboswitches as model systems. This proposal details a set of interconnected specific aims that addresses fundamental questions related to these research goals: (1) what is the global structure of purine- sensing riboswitches, (2) why do purines inefficiently regulate the THF riboswitch, and (3) how do cobalamin riboswitches functionally couple effector binding to gene regulation? To address these questions, a combination of structural (x-ray crystallography) biophysical (calorimetry and stopped-flow kinetics), biochemical (chemical footprinting), genetic and molecular biological (in vivo and in vitro activity assays) and bioinformatics/computational approaches will be combined to study the structure-regulatory activity linkage in riboswitches. A deeper knowledge of how RNA specifically interacts with small molecules and affect its structure and activity will help pav the way for a new generation of therapeutics that target non-protein coding RNAs in bacteria and eukaryotes.